8) and by electrophoresis. First-strand complementary DNA was synthesized using equal amounts (0.5 ��g) of total RNA, with the SuperScript VILO complementary DNA synthesis kit (Invitrogen, Carlsbad, CA). The messenger RNA (mRNA) levels were analyzed by quantitative real-time PCR (qRT-PCR) with SYBR Green chemistry (Fluocycle II SYBR green mix; Euroclone, Pero, Italy). All the reactions were performed in triplicate with the Opticon2 qRT-PCR system (MJ Research, Waltham, MA). In patients with and without NAFLD, we evaluated the PNPLA3/adiponutrin mRNA levels, and we correlated the PNPLA3 I148M genotype with the expression of insulin receptor (INSR), which regulates insulin signaling and is down-regulated in the Anti-infection Compound Library solubility dmso metabolic syndrome,27 selleckchem steroid regulatory element binding protein 1c (SREBP1c), which regulates lipogenesis,28 peroxisome proliferator-activated receptor-�� (PPAR-��), which regulates lipolysis and is decreased in NASH,29 and Fas ligand (FASL), which is a proapoptotic molecule induced by insulin resistance.30, 31 Primers are shown in Table 2. Results were normalized for ��-actin and 18S RNA, which were chosen as a control because of stable expression among different samples. Results are expressed as means �� standard deviation. Mean values were compared by analysis of variance (ANOVA) and post-hoc analysis or Wilcoxon test, when appropriate, and frequencies by Fisher's exact test, and chi-squared test for trend, when appropriate. Variables were correlated by the Spearman's rho test. The association between the I148M variant and the presence of metabolic abnormalities, NASH, and fibrosis was evaluated by multivariate logistic regression analysis. In analyzing the association between I148M and steatosis and fibrosis, we compared allele and genotype frequencies in those with none or mild steatosis/fibrosis (0 and 1/3 for steatosis; ATP7A 0 and 1/4 for fibrosis) versus those with moderate/severe steatosis/fibrosis (2 and 3/3 for steatosis; 2, 3, and 4/4 for fibrosis). Analyses were carried out with JMP 6.0 statistical analysis software (SAS Institute Inc, Cary, NC). The frequency distribution of the rs738409 CG adiponutrin genotype, which was in Hardy-Weinberg equilibrium in Italian and UK patients and in controls, is shown in Table 3. The frequency distribution of the mutant G allele was significantly higher in Italian patients with NAFLD than in geographically-matched, age-matched, and sex-matched healthy controls with normal liver enzymes and metabolic parameters and a normal fatty liver index (P < 0.0001; Table 2).
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